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Patel, Muneeza S. "Algorithms for E. coli genome engineering." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/106461.
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Thesis: M. Eng. in Computer Science & Molecular Biology, Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
"June 2016." Page 90 blank. Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 70-72).
Author summary: Lamba red recombineering is one of methods of performing genome engineering. However, this method of genome editing is not very specific and efficient and is highly dependent on the genomic regions that are targeted (integration sites). In this project we explored ways of identifying what makes a site well suited for lambda red genome engineering. We wanted to explore whether we can eventually predict the "goodness" of an integration site using an algorithm. Our initial approach to the problem was to write an algorithm based on some characteristics that we felt would be key to determining the goodness of a site. Choosing to initially focus on specificity of the integrations, we used experimental approaches to evaluate whether our algorithm had any predictive powers for specificity. Upon failing, we revised our plan to generate a dataset of ~150 sites and their integration data (whether integration was successful, specific and efficient at that site). We used this dataset to explore correlations between the specificity data and characteristics we thought might affect the specificity of sites. The most promising characteristics appeared to be the uniqueness of the genomic site (as determined by BLAST) and the existence of Repetitive Extragenic Palindrome (REP) sites at the site of integration. Section I of this thesis sets up the problem, section II talks about the initial approach we took to the problem and section III discusses our modified approach -- which formed the bulk of this thesis project. Section I and III are the most relevant to understand the project, while Section II gives more content to the project in addition to detailed insight to what approaches did not work.
by Muneeza S. Patel.
M. Eng. in Computer Science & Molecular Biology
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Neelakanta, Girish. "Genome variations in commensal and pathogenic E.coli." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974330329.
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Schlegel, Susan. "From protein production to genome evolution in Escherichia coli." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-94993.
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The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields. To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.
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Wlodarski, Michal. "Dynamics of E. coli genome and cytosol under antibiotics." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275205.
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In light of an urgent need for improved antimicrobial diagnostics and therapeutics, understanding bacterial behaviour, and bacterial responses to treatments in particular, is one of the key objectives of modern medical research. While the molecular mode of action of antibiotics is usually well known, their effect on the cell at a "systems" level (on the regulatory networks, metabolism, etc.) is only beginning to be quantitatively understood. We address some of these response phenotypes in Escherichia coli testing different antibiotic classes and growth conditions. We study the short (< 15 s) time-scale fluctuation dynamics of fluorescently-tagged chromosomal loci and cytosolic aggregates, which report for the state of locus ”compaction” and the levels of macromolecular crowding of the cytosol, respectively. We improve the precision of those measurements developing a novel data treatment procedure and discover that sub-lethal doses of ciprofloxacin, rifampicin, and vancomycin as well as hyperosmotic shock conditions cause small but consistent changes (unique to each treatment agent) to the physical organisation of chromosomal Ori2 and Ter3 loci and the cytosol. We reveal, among other findings, strong correlations between the effects in different parts of the chromosome and between the chromosome and cytosol. In addition, we complement the marker dynamics work with single-cell level gene expression measurements during sub-lethal translation inhibition. Specifically, we compare responses to tetracycline and chloramphenicol from constitutive and ribosomal promoters in Ori3 and Ter3 chromosomal positions over long (7 h) treatment times in exponentially growing bacteria. We reveal, for the first time, the kinetics of cellular resource allocation and provide novel insights on globally regulated transcription, relevant to the three-component proteome partitioning model, gene-length dependent effects of the processivity of translation, and ”reversibility” of ribosome-binding antibiotics. In addition, we discover a strong correlation between the timing of responses from promoters in the Ori3 and Ter3 positions, and a small but consistent difference in the response magnitude between the two positions.
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Romero, Alvarez David. "Genome wide analyses of the Escherichia coli primary and secondary transcriptomes." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/6917/.
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Escherichia coli K12 serves as an important model for studying systems that are important to bacteria in their own right as well as those that are conserved in ‘higher' organisms, which are more difficult and costly to study. Like many model organisms, the genome of K12 has been sequenced, producing a catalogue of protein-coding and stable-RNA genes that enabled study using ‘omic’ approaches. This has led to a rapid expansion of our knowledge of patterns of gene expression and their dependency on growth conditions, cell physiology and individual genes. However, the underlying networks of gene regulation are less well understood, but are known to involve the control of steps in RNA processing and degradation as well as transcription and translation. With this in mind, this thesis describes the development of an approach based on RNA sequencing that produces nucleotide-resolution transcriptome maps that distinguish sites that correspond to RNA processing and steps in degradation from those of transcription initiation, while incorporating all classes of RNA. Comparison with results obtained previously validated the approach, which has been applied already to the study of other bacterial species. Within the E. coli map, many new features were identified, such as previously undetected small RNAs and processing at a site associated with the production of specialised ribosomes, which may ensure the translation of leaderless mRNAs, which were also mapped. The approach also showed the benefit of incorporating steps that can differentiate the 5’ status of transcripts in assigning sites of transcription initiation. RNA sequencing was also used to map sites of cleavage by RNase E, an essential endoribonuclease that is central to both the processing and degradation of RNA in bacteria and plant plastids. This aspect of the thesis has advanced from pilot studies to the point where the ‘code’ that determines one form of substrate recognition by RNase E is beginning to emerge. As a result of this success, equivalent data has been collected for other ribonucleases involved in RNA processing and degradation. Continuing analysis of the primary and secondary transcriptomes, consisting of native, unprocessed transcripts and of transcripts that have been modified from their native form via processing and/or degradation respectively, with the tools presented here promises to broaden and deepen our understanding of an important model organism.
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Coss, Dennis. "Insertion of genes and operons into the Escherichia coli genome through targeted recombination." Morgantown, W. Va. : [West Virginia University Libraries], 2005. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=3804.
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Thesis (Ph. D.)--West Virginia University, 2005.Title from document title page. Document formatted into pages; contains v, 125 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 71-87).
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Mosberg, Joshua Adam Weintrob. "Studying and Improving Lambda Red Recombination for Genome Engineering in Escherichia coli." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10777.
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The phage-derived Lambda Red recombination system utilizes exogenous DNA in order to generate precise insertion, deletion, and point mutations in Escherichia coli and other bacteria. Due to its convenience, it is a frequently-used tool in genetics and molecular biology, as well as in larger-scale genome engineering projects. However, limited recombination frequency constrains the usefulness of Lambda Red for several important applications. In this work, I utilize a mechanism-guided approach in order to improve the power and utility of Lambda Red recombination.
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El, Sayyed Hafez. "Mapping Topoisomerase IV Binding and Activity Sites on the E. coli genome." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS362/document.
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Des liens de caténation sont progressivement crées lors de la réplication de l’ADN et sont responsables de la cohésion des chromatides sœurs. La topoisomérase IV est une topoisomérase de type II impliquée dans la résolution de ces liens de caténation accumulés derrière la fourche de réplication, et lors de la dernière étape de séparation des chromatides sœurs à la fin de la réplication. Nous avons étudié la liaison de la topoIV à l’ADN ainsi que son activité catalytique à l’aide de méthodes de biologie moléculaire et de génomique. Une expérience de ChIPseq a révélé que l’interaction de la topoIV de chez E.coli avec l’ADN est contrôlée par la réplication. Durant la réplication, la topoIV a accès à des centaines de sites sur l’ADN mais ne se lie qu’à quelques sites où elle exerce son activité catalytique. La conformation locale de la chromatine et l’expression des gènes influencent la sélection de certains sites. De plus, une forte liaison et une activité catalytique renforcée a été trouvée au site de résolution des dimers, dif. Le site dif est situé à l’opposé de l’origine de réplication dans le macrodomaine ter. Nous avons montré qu’il existe une interaction physique et fonctionnelle entre la topoIV et la recombinase XerCD, qui agit au site dif. Cette interaction est médiée par MatP, une protéine essentielle dans l’organisation du macrodomaine ter. L’ensemble de ces résultats montre que la topoIV, XerCD/dif et MatP œuvrent ensemble pour permettre l’étape finale de ségrégation des chromosomes lors du cycle cellulaireCatenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle
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Johnson, Matthew David. "Understanding the regulation of acid resistance in E. coli using whole genome techniques." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/3006/.
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The ability of bacteria to thrive in a variety of host environments depends on their capacity to sense and respond to a wide array of stressors. E. coli encounters many stresses during transit through the gastro-intestinal tract, including acid stress. Acid stress response in E. coli is regulated by a complex network called AR2. The AR2 network comprises several local regulators that collate signals from multiple two-component systems (TCS) including RcsBD, EvgAS and PhoPQ. We combined lab-based evolution and whole genome re-sequencing to generate and identify mutations that confer increased acid resistance in E. coli K-12. All of these mutations map in the gene encoding EvgS, the sensor kinase of the EvgAS TCS. Using a luciferase reporter system and phenotypic assays we characterised the nature of these evgS mutations and their contribution to acid resistance. We also used high-temporal resolution luciferase reporter assays to uncover novel aspects of this network and implicate PhoP in the repression of acid resistance. Finally, we used our evgS mutants to characterise novel interactions within the AR2 network between the two component systems RcsBD and EvgAS. These results are discussed in relation to the role of regulatory networks in bacteria.
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Schmidt, Dorothea. "Molekulare Analyse des probiotischen Stamms Escherichia coli Nissle 1917." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1243973355362-88295.
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Der probiotische Stamm E. coli Nissle 1917 ist ein Fäkalisolat, das in der Medizin traditionell zur Behandlung verschiedener gastrointestinaler Erkrankungen eingesetzt wird. Durch erfolgversprechende klinische Studien zur Remissionserhaltung bei Colitis ulcerosa, bei denen EcN als therapeutische Alternative zur Standardmedikation eingesetzt wird, ist das Interesse an den Wirkmechanismen von Probiotika stark gestiegen. EcN gehört derzeit zu den am besten untersuchten Probiotika. Einige Wirkmechanismen konnten dadurch schon aufgeklärt werden. So sind vermutlich Strukturkomponenten und stammspezifische Syntheseleistungen an der Ausprägung des probiotischen Phänotyps von EcN beteiligt. Schlüssige Konzepte, die über Gene, Genprodukte und molekulare Mechanismen den probiotischen Effekt von EcN erklären, fehlen bislang. Im Rahmen dieser Arbeit wird das Genom von EcN analysiert und auf der Basis der Genomsequenz mit anderen E. coli-Stämmen verglichen. Mit Hilfe einer Promotor-Reporter-Fusionsbibliothek (Promotorbank) werden intestinal in vivo regulierte Gene identifiziert und dadurch neue Ansätze zur Untersuchung der probiotischen Eigenschaften von EcN geschaffen. Die Grundlage für die molekulare Analyse von EcN ist die manuelle Nachannotation seines sequenzierten Genoms. Die EcN-Sequenz wird mit 13 weiteren annotierten E. coli-Sequenzen verglichen. Nach dieser Analyse kodiert EcN derzeit 121 stammspezifische Gene. Die Genomstruktur ist mit den enthaltenen genomischen Inseln und Prophagen dem Genom des uropathogenen E. coli CFT073 sehr ähnlich. Mit wenigen Ausnahmen kodiert EcN alle in E. coli CFT073 vorhandenen Virulenz- und Fitnessfaktoren, so dass auf der Nukleotidebene die nahe Verwandschaft dieser beiden Stämme bestätigt werden kann. Zudem kann gezeigt werden, dass EcN in artifiziellen Systemen wie der Zellkultur oder gnotobiotischen Mäusen ein pathogenes Potenzial hat, obgleich die Kolonisierungsfähigkeit pathogener Bakterien durch Inkubation mit EcN herabgesetzt wird. Eine wichtige Rolle bei der Besiedlung des Intestinaltrakts und der Immunstimulation von Darmepithelzellen spielt auch die globale Regulation der Genaktivität bei EcN durch den alternativen Sigma-Faktor RpoS, der im Gegensatz zu rpoS-Deletionsmutanten zu einer gesteigerten mRNA-Expression des Tight-junction Proteins ZO-1 führt. Des Weiteren führte die Untersuchung von EcN-Deletionsmutanten zu der Schlussfolgerung, dass einige genomische Inseln für Eigenschaften, die das probiotische Verhalten erklären können, eine Rolle spielen. Durch den Einsatz einer Promotorbank von EcN in konventionellen und gnotobiotischen Mäusen werden erstmalig Sequenzen von intestinal in vivo aktiven Promotoren identifiziert. Der Aufbau eines Promotor-Reportergen-Assays mit dem Biolumineszenz erzeugenden luxCDABE-Operon ermöglichte die Untersuchung ausgewählter Promotoren in vitro. Mit einem In Vivo Imaging System (IVIS) kann in weiteren Experimenten die Aktivität dieser Promotoren in lebenden Mäusen untersucht werden. Im Rahmen dieser Arbeit wird gezeigt, dass EcN kein vollkommen harmloser probiotischer Stamm ist. Weitere Informationen über EcN sind dehalb wichtig für eine optimierte Anwendung als Therapeutikum. Die molekulare Analyse ist somit eine unbedingt notwendige Grundlage für weiterführende Untersuchungen der Eigenschaften von EcN, die für seinen probiotischen Charakter verantwortlich sindThe probiotic E. coli Nissle 1917 is a fecal isolate which is traditionally used for treatment of various gastrointestinal disorders. In clinical trials where EcN was used as therapeutic alternative for remission maintenance of ulcerative colitis compared to standard medication, promising results led to an increased interest in probiotics. Today, EcN is one of the best studied probiotics. Therefore, several mechanisms of action could be enlightened. Structural components and strain-specific products are responsible for its probiotic effects. But conclusive concepts about genes, gene products and molecular mechanisms that really contribute to the probiotic character of EcN have not been offered so far. In order to create new possibilities to elucidate the probiotic traits of EcN the genome is analysed by taking this as a basis for comparison to other E. coli genomes and identification of intestinal in vivo regulated genes using a promoter-trap-library. The sequenced EcN genome is annotated and compared to 13 other so far annotated E. coli genomes. Concerning these analyses EcN encodes 121 strain-specific genes. The genome structure including the genomic islands and prophages is highly homolog to the uropathogenic E. coli CFT073. EcN encodes most of the virulence and fitness factors that are present in E. coli CFT073. Therefore, the close relationship of these two strains is confirmed at nucleotide level. Furthermore, it is shown that in artificial systems like cell culture assays and gnotobiotic mice EcN reveals a pathogenic potential although EcN is able to decrease colonization efficiency of pathogenic bacteria. The alternative sigma factor RpoS that is responsible for global regulation and activity of several genes seems to play an important role during colonization of EcN in the intestine and its immunostimulatory effects on intestinal epithelial cells. Investigation of EcN-deletion mutants lacking genomic islands and prophages lead to the conclusion that some genomic islands may play a role for specific probiotic traits. This is the first time where a promoter-trap-library was used in conventional and gnotobiotic mice for collection of intestinal in vivo active promoters. Constructing and establishing a promoter-reporter gene assay with the bioluminescent luxCDABE operon made the investigation of selected promoters in vitro possible as well as establishing a bioluminescence assay using an In Vivo Imaging System (IVIS) for investigation of promoter activity in living mice. In this research project was shown that EcN is not a completely harmless probiotic. The genome structure and regulatory mechanisms of gene expression are the strain’s molecular traits that lead to probiotic activity and immunostimulatory effects. Therefore, the molecular analyses presented here, together with the complete genome sequence, are a basis for further investigations of mechanisms that are responsible for the probiotic effects of EcN
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Coulange, Frédérique. "Isolement et caracterisation de regions specifiques du genome des escherichia coli pathogenes aviaires (doctorat : microbiologie)." Paris 11, 1999. http://www.theses.fr/1999PA114802.
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Brambilla, Elisa. "Investigation of E. coli genome complexity by means of fluorescent reporters of gene expression." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066607/document.
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Escherichia coli est capable de survivre dans de nombreux environnements différents. Les informations nécessaires à cette adaptation sont codées dans le chromosome. Cette molécule circulaire est condensé dans une structure compacte protéines-ADN, appelée nucléoïde. Le chromosome n¿est pas uniforme et montre notamment une distribution inégale de sites de fixation de protéines et de séquences riches en AT. Il a été montré que la position des gènes importants pour la cellule est hautement conservée dans les gamma-protéobactéries. Ces différences le long du chromosome et cette conservation de la position suggèrent que la position du gène peut influencer son expression. Pour tester cette hypothèse, on a étudié l'expression d'un gène fluorescent inséré dans différentes positions autour du chromosome. L'expression de ce gène est contrôlé par des promoteurs différemment régulés: un est réprimé par la protéine H-NS, un est non régulé et un est sensible au superenroulement de l'ADN. Nous avons étudié l'expression dynamique de ces promoteurs pendant les différentes phases de croissance dans différentes conditions. Nous avons montré que l'expression du promoteur dépendant de la protéine H-NS est liée à l'emplacement sur le chromosome. En effet, la répression par H-NS est accrue en présence de séquences riches en AT. Nous avons également étudié l'influence d'un gène divergent sur l'expression de gènes rapporteurs en fonction de la position chromosomique. Nous avons montré que cette influence dépend de la localisation du gène. Nous avons donc demontré l'impact de la position chromosomique sur l'expression des gènes tout en donnant une nouvelle perspective sur la complexité du génomeEscherichia coli is able to survive in many different environments. The information necessary for this adaptation is encoded in the chromosome. This circular molecule is condensed in a compact DNA-protein structure, called the nucleoid. The chromosome is not uniform, and shows uneven distributions of nucleoid-associated proteins (NAPs) binding sites, AT-rich sequences and general protein occupancy domains. It has been demonstrated that the position of important genes is highly conserved in ?-Proteobacteria. These differences along the chromosome and the conserved position of important genes suggest that the position of the gene can influence gene expression. To test this hypothesis, I studied the expression of a fluorescent reporter gene inserted in different positions around the chromosome. The expression of the reporter is driven by differently regulated promoters, one repressed by the important NAP H-NS, one non regulated and one subject to supercoiling and stringent control. We studied the dynamical expression of these promoters in different growth conditions, growth phases, upon nutritional upshift and under stress. We showed that the expression of the H-NS dependent promoter depends on the location on the chromosome, because H-NS repression is enhanced in presence of AT-rich sequences. We also studied the influence of a divergent gene on the reporter expression as a function of chromosomal position, and showed that this influence depends on the location of the gene. With our study we have been therefore able to show the impact of chromosomal position on gene expression and to give a new perspective on genome complexity
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PRADEL, NATHALIE. "Escherichia coli producteurs de shiga-toxines : etude epidemiologique, recherche des caracteristiques des souches pathogenes par comparaison moleculaire et hybridation soustractive (doctorat : microbiologie)." Clermont-Ferrand 1, 2001. http://www.theses.fr/2001CLF1PP02.
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Médigue, Claudine. "Conception et exploitation d'une base de donnees specialisee dediee a l'analyse du genome d'escherichia coli." Paris 6, 1991. http://www.theses.fr/1991PA066690.
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L'exploitation des donnees de sequences acquises au cours du sequencage de genomes cellulaires entiers suppose la construction de base de donnees integrant a la fois les donnees brutes issues du sequencage et l'expertise des biologistes a propos de chacun des genes repertories. Le travail presente dans cette these a pour objectif l'elaboration d'un environnement informatique permettant d'organiser, de gerer et d'exploiter un ensemble de donnees biologiques issues d'un meme organisme. Dans un premier temps, le developpement d'une base de donnees specialisee autour du genome d'escherichia coli nous a conduit a definir un modele de structuration des donnees permettant de prendre en compte des connaissances jusqu'a present mal structurees relatives a l'organisation du chromosome bacterien. Ce modele devrait etre applicable a l'etude d'autres organismes procaryotes. Nous avons par ailleurs mis en uvre diverses methodes informatiques permettant d'exploiter la base de donnees. Les resultats biologiques que nous decrivons concernent l'analyse des donnees de la carte genetique et physique d'e. Coli et la caracterisation des genes connus de la bacterie repertories dans la base en fonction de leur utilisation des codons. Trois classes de genes ont ainsi ete identifiees qui se distinguent a la fois par leur fonction et par leur structure. En particulier l'une de ces classes est essentiellement composee de genes impliques dans les echanges horizontaux. Ce resultat nous a conduit a analyser dans le detail les proprietes specifiques des classes de genes d'e. Coli qui sont comparees a celles que nous obtenons pour b. Subtilis et s. Cerevisiae. Ces etudes devraient nous permettre de caracteriser les contraintes specifiques qui agissent sur les sequences nucleotidiques et ainsi de mieux comprendre l'organisation du texte genomique de chacun de ces organismes
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Henze, Stefanie [Verfasser], and Claudia [Akademischer Betreuer] Acquisti. "The impact of nitrogen deprivation on genome evolution in Escherichia coli / Stefanie Henze ; Betreuer: Claudia Acquisti." Münster : Universitäts- und Landesbibliothek Münster, 2016. http://d-nb.info/1141682036/34.
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Cowley, Lauren A. "Use of genome sequencing to investigate the molecular basis of bacteriaphage interaction of the Escherichia coli O157 typing phages and the elucidation of the biological and public health significance of phage type." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23583.
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Background Shiga toxin producing Escherichia coli (STEC) O157 causes severe gastrointestinal disease and haemolytic uremic syndrome, and has a major impact on public health worldwide with regular outbreaks and sporadic infection. Phage typing, i.e. the susceptibility of STEC O157 strains to a bank of 16 bacteriophages, has been used in the UK to differentiate STEC O157 for the past 25 years and the phage type (PT) can be an epidemiological marker of strains associated with severe disease or associated with cases that occur from foreign travel. However, little is known about the molecular interactions between the typing phages (TP) and STEC O157. The aims of this thesis were to use whole genome sequencing to elucidate the genetic basis for phage typing of STEC O157 and through this understand genetic differences between strains relevant to disease severity and epidemiology. Results Sequencing the STEC O157 TPs revealed that they were clustered into 4 groups based on sequence similarity that corresponded with their infectivity. Long read sequencing revealed microevolutionary events occuring in STEC O157 genomes over a short time period (approximately 1 year), evidenced by the loss and gain of prophage regions and plasmids. An IncHI2 plasmid was found responsible for a change in Phage Type (PT) from PT8 to PT54 during two related outbreaks at the same restaurant. These changes resulted in a strain (PT54) that was fitter under certain growth conditions and associated with a much larger outbreak (140 as opposed to 4 cases). TraDIS (Transposon directed Insertion site sequencing) was used to identify 114 genes associated with phage sensitivity and 44 genes involved in phage resistance, emphasising the complex nature of identifying specific genetic markers of phage susceptibility or resistance. Further work is required to prove their phage-related functions but several are likely to encode novel phage receptors. Deletion of a Stx2a prophage from a PT21/28 strain led to a strain that typed as PT32, supporting the concept that the highly pathogenic PT21/28 lineage I strains emerged from Stx2c+ PT32 strains in the last two decades by acquisition of Stx2a-encoding prophages. Conclusions This body of work has highlighted the complexity of bacteriophage interaction and investigated the genetic basis for susceptibility and resistance in E. coli. The grouping of the TPs showed that resistance or susceptibility to all members of a typing group was likely to be caused by one mechanism. IncHI2 was identified as one of the markers for the PT54 phenotype. The Stx2a prophage region was associated with the switch from PT32 to PT21/28, although PT32 strains containing both Stx2a and Stx2c-encoding prophages have been isolated and can provide insights into phage variation underpinning the susceptibility to the relevant typing phages. The TraDIS results indicated that susceptibility or resistance was governed by multiple genetic factors and not controlled by a single gene. The significance of LPS for initial protection from phage adsorption was evident and a number of novel genes controlling phage susceptibility and resistance identified including the Sap operon and stringent starvation protein A respectively. While SNP-based typing provides an excellent indication of the evolution and relatedness of strains, phage typing can provide real insights into short term evolution of the bacteria as PTs can be altered by mobile elements such as prophages and plasmids. This study has shown that, although complex, genetic determinants for PT can be mined from the genome and allow us to understand the evolution of this zoonotic pathogen between host species and during outbreaks.
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Lember, Geivi. "Sepsis-associated Escherichia coli whole-genome sequencing analysis using in-house developed pipeline and 1928 diagnostics tool." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-19841.
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Sepsis is a life-threatening condition that is caused by a dysregulated host response to infection. Timely detection of sepsis and antibiotic treatment is important for the patient’s recovery from sepsis. Usually, when sepsis is detected, immediate antibiotic treatment is started with broad-spectrum antibiotics as it takes time to determine the correct antibiotic susceptibility. To overcome this problem, next-generation sequencing is seen as one possible development in clinical diagnostics in the future. Automated bioinformatics pipelines could be used initially for surveillance purposes but eventually for rapid clinical diagnosis. Therefore, the results of 1928 Diagnostics, an automated pipeline for whole-genome sequencing (WGS) data analysis, were compared with the results of an in-house developed pipeline for manual data processing by analyzing sepsis-associated Escherichia coli (SEPEC) WGS data. The pipelines were compared by assessing their predicted antimicrobial resistance (AMR) genes, virulence genes and epidemiological relatedness. In addition, the predicted resistance genes were compared to phenotypic antimicrobial susceptibility testing (AST) data from the clinical microbiology laboratory. All the results obtained from the 1928 Diagnostics and in-house pipeline were similar but differed in the number of virulence/predicted AMR genes, AMR gene variants, detection of species and epidemiologically related E. coli samples. Moreover, the predicted AMR genes from both pipelines did not show a good overall relation to the phenotypic AST result. More studies are needed to make predictions of genes from the WGS analysis more reliable so that WGS analysis can be used as a diagnostics tool in clinical laboratories in the future.
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Ma, Chih-Yu. "Occurrence and characterization of antibiotic-resistant Escherichia coli in wastewater and surface water." Kyoto University, 2020. http://hdl.handle.net/2433/259030.
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CHEVALLIER, BRUNO. "Structure et expression du genome de lactobacillus plantarum : clonage et etude de la stabilite de l'adn chez escherichia coli." Université Louis Pasteur (Strasbourg) (1971-2008), 1994. http://www.theses.fr/1994STR13184.
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Afin de cloner et d'etudier les genes intervenant dans la voie de biosynthese des pyrimidines d'une bacterie lactique, lactobacillus plantarum, nous avons entrepris la construction d'une banque d'adn genomique de l. Plantarum dans un vecteur navette escherichia coli-bacterie gram positive chez e. Coli. Nous avons commence par estimer, par electrophorese en champ pulse, en utilisant les endonucleases asci, sfii et i-ceui, la taille du genome de 8 souches de l. Plantarum et obtenu des valeurs comprises entre 3 a 4,2 mb. Au moins 5 loci codant pour les arn ribosomiques ont ete mis en evidence dans le genome de ces souches. Les profils de restriction et de rapd (random amplified polymorphic dna) du genome obtenus se sont reveles etre des outils taxinomiques interessants. Nous avons mis en evidence une instabilite de l'adn genomique de l. Plantarum clone dans plusieurs plasmides (pam239, pdl278, pnz12, ptg262, puc19) chez e. Coli. Une faible proportion de clones recombinants et une majorite de plasmides deletes sont les deux caracteristiques de cette instabilite. L'utilisation de differents plasmides caracterises par des terminateurs de transcription presente de chaque cote du site de clonage ou un faible nombre de copies par cellule, ne reduit pas cette instabilite ; par contre la methylation dam de l'adn a une influence. L'instabilite de l'adn genomique de l. Plantarum, independante des systemes de recombinaison homologue, est probablement due, chez e. Coli, a un mecanisme de recombinaison illegitime encore inconnu. L'efficacite de transformation par electroporation de l. Plantarum ccm 1904 a ete amelioree de 100 a 1000 fois de maniere a obtenir 100 000 transformants par microgramme d'adn plasmidique. L. Plantarum ccm 1904 se transforme 5 a 30 fois mieux par un plasmide non methyle dam. L'ensemble des resultats obtenus nous permet d'esperer construire une banque d'adn genomique chez l. Plantarum ou cloner directement les genes par complementation des mutants de cette espece obtenus au laboratoire. Nous eviterons ainsi l'instabilite de l'adn genomique de l. Plantarum observee chez e. Coli
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Gunasekera, Samantha Thilini. "Evaluating whole-genome sequencing strategies in AMR research through characterisation of mobile genetic elements in wildlife-origin Escherichia coli." Thesis, Gunasekera, Samantha Thilini (2019) Evaluating whole-genome sequencing strategies in AMR research through characterisation of mobile genetic elements in wildlife-origin Escherichia coli. Honours thesis, Murdoch University, 2019. https://researchrepository.murdoch.edu.au/id/eprint/50510/.
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Background
Resistance against antimicrobials critically important to human medicine has been frequently observed in livestock and clinical settings where the selection pressure imposed by antimicrobial use is high. Today, it is detected in wildlife even in the absence of a direct selection pressure. On Penguin Island, 660 m from the coast of Western Australia, Escherichia coli isolated from little penguin and feral pigeon faecal samples (n = 20) was found to be phenotypically resistant to extended-spectrum cephalosporins and fluoroquinolones. It was hypothesised that the frequent observations of resistance against CIAs in this group of birds were a result of horizontal gene transfer. However, mobile genetic elements have historically been challenging to characterise using second-generation sequencing technology alone. These difficulties have been linked to coverage bias caused by library preparation, and to insufficient read length to assemble long, repetitive genomic regions. To address the limitations of read length, second-generation sequencing technologies were used in conjunction with third-generation sequencing technologies to produce more contiguous assemblies while maintaining accuracy. To address coverage bias, the performance of the Nextera XT and Nextera Flex library preparation kits were evaluated to assess whether either library preparation kit was associated with reduced sequencing bias.
Results
The results of the library preparation kit comparison found that the Nextera Flex library preparation kit produced more even coverage than the Nextera XT kit, however tagmentation bias, GC content bias, de novo assembly quality and antimicrobial resistance (AMR) gene detection was either unchanged or poorer than Nextera XT.
Resistance to extended-spectrum cephalosporins observed in the E. coli isolates was mediated mainly by blaCTX-M-15 and was shown to have circulated through the different isolates via mobile genetic elements such as ISEcp1, IS26 and Tn2. Resistance against fluoroquinolones occurred primarily through mutations in quinolone resistance-determining regions, with only a minority of isolates harbouring plasmid-mediated quinolone resistance genes. Using a combination of second and third generation sequencing technology, a composite transposon conferring multidrug-resistance to macrolides, folate pathway inhibitors and aminoglycosides was found in varying conformations in over half of the E. coli isolates.
Conclusions
This study was the first to assess the Nextera Flex library preparation kit in the context of AMR research and found that coverage bias was markedly improved. However, this did not impact the practical applications of whole-genome sequencing data such as de novo assembly or AMR gene detection.
Using a combination of second and third-generation sequencing technologies, this study established the presence of multiple mobile genetic elements conferring resistance to critically important antimicrobials in E. coli isolated from avian wildlife on Penguin Island, Western Australia.
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Nguyen, Huong LE. "Etude des facteurs régulateurs de la traduction chez Escherichia coli." Thesis, Toulouse, INSA, 2019. http://www.theses.fr/2019ISAT0004.
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L’analyse des régulations de l’expression des gènes chez les bactéries permet de comprendre l’adaptation des bactéries à leur environnement et dans un contexte de biologie de synthèse d’optimiser la production microbienne de molécules d’intérêt. Notre objectif a été d’étudier la traduction au niveau du génome et ses relations avec les autres processus cellulaires par une approche de biologie des systèmes. Le traductome a été mesuré : pour chacun des ARN messagers, son pourcentage de copies en traduction et sa densité en ribosomes. Pour la première fois, une image complète de l’état traductionnel de E. coli en croissance rapide a été obtenue, caractérisée par une majorité de transcrits avec un très fort pourcentage de copies en traduction mais faiblement chargés en ribosomes. Notre modèle statistique a identifié des facteurs liés à la séquence comme déterminants de la traduction et le rôle important d’un paramètre physiologique : la concentration en ARNm. Pour la première fois, cet effet de la transcription sur la traduction a été validé à l’échelle moléculaire sur plusieurs gènes. Nous avons montré qu’une augmentation de la concentration d’un ARNm par induction transcriptionnelle entrainait une augmentation du pourcentage de copies en traduction et de la charge en ribosomesThe analysis of gene expression regulation is necessary to better understand bacterial adaptation to environment and to be able in a context of synthetic biology to optimize the production of molecules of interest. The goal of this thesis was to study translation at the genome-wide level and its relationship to other cellular processes using a systems biology approach. First, translation activity at the -omic scale (called the traductome) was measured : for each messenger RNAs, its percentage of copies in translation and ribosome density. For the first time, a complete picture of the translational state in fast growing E. coli cells was obtained, characterized by a majority of transcripts with a very high percentage of copies in translation but a low ribosome density. Our model identified sequence-related factors as determinants of translation but, more surprisingly, the model predicted the important role of a physiological parameter: the mRNA concentration. Thus, more concentrated mRNA would have higher percentage of copies in translation and higher ribosome density. For the first time, this effect of transcription on translation has been validated at the molecular level on several genes. We showed that an increase in mRNA concentration by transcriptional induction results in increases in percentage of copies in translation and in ribosome load
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Raeside, Colin. "Plasticité du génome au cours d'une expérience d'évolution au long terme chez Escherichia Coli." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV070/document.
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Les réarrangements d'ADN à grande échelle, tels que inversions, amplifications, duplications, délétions, insertions et transposition des éléments génétiques mobiles, sont des acteurs essentiels de l'évolution. Ils ont une forte incidence sur l'organisation et l'expression des chromosomes, ce qui affecte le phénotype des organismes. Toutefois, la dynamique de ces réarrangements au cours de l'évolution et leurs effets sur l'adaptation des organismes sont souvent inconnus. Nous avons abordé ces questions en utilisant la plus longue expérience d'évolution en cours. A partir d'un ancêtre commun d'Escherichia coli, douze populations indépendantes sont cultivées dans un milieu limité en glucose depuis plus de 60 000 générations, soit 26 ans. La plupart des études antérieures ont porté sur les mutations ponctuelles et les petites insertions et délétions (InDels). En utilisant des clones isolés au cours du temps dans ces 12 populations, nous avons caractérisé les réarrangements d'ADN à grande échelle à la fois par l'analyse des séquences de génomes et par cartographie optique. A 40 000 générations, nous avons identifié 110 réarrangements parmi lesquelles 82 délétions, 19 inversions et 9 duplications. Plusieurs régions du chromosome ont été touchées à plusieurs reprises par le même type de réarrangements dans des populations indépendantes. Dans une des populations au moins, les réarrangements se sont produits au début de l'expérience d'évolution, au moment où l'augmentation de la valeur sélective est la plus élevée. Par conséquent, certains de ces réarrangements pourraient être bénéfiques dans ces conditions. Même dans le cas contraire, nous avons montré que ces réarrangements affectaient fortement la structure du chromosome au cours de l'expérience d'évolution.Au niveau moléculaire, nous avons montré que ~ 70% des réarrangements se produisent par recombinaison entre séquences d'insertion (IS), ce qui illustre l'importance de ces dernières dans la plasticité du génome. Nous avons donc caractérisé la distribution et la dynamique de ces petits éléments génétiques mobiles dans l'ensemble des 12 populations. Nous avons montré que les éléments IS ont fortement contribué à l'ensemble des mutations après 40 000 générations. Dans une population, les IS représentent même la moitié des mutations, et un des types d'IS, IS150, présente une forte prolifération avec 6 fois plus de copies à 40 000 générations, intervenant dans la plupart des réarrangements détectés dans cette population. Nous avons montré une forte dynamique temporelle d'IS150, avec une forte expansion suivie d'une domestication par l'hôte. En testant trois scenarii évolutifs, nous avons démontré que l'expansion d'IS150 était liée à une forte augmentation de la valeur sélective conférée par les événements initiaux de transposition ayant eu lieu avant 2000 générations. Plus tard, entre 20 000 et 40 000 générations, nous avons mesuré une diminution de la fréquence de transposition, probablement en raison d'une régulation négative de la transposition imposée par l'hôte. Enfin, et pour la première fois, nous avons développé un modèle d'évolution de la dynamique des IS, qui confirme que leur expansion est liée à un nombre seuil d'insertions bénéfiques initiales. Ces résultats montrent que les réarrangements chromosomiques à grande échelle et les éléments IS ont joué un rôle actif dans la trajectoire évolutive au cours de 40 000 générations d'évolution bactérienneLarge-scale DNA rearrangements, including inversions, amplifications, duplications, deletions, insertions, and transposition of mobile genetic elements, are major drivers of evolution and strongly impact on chromosome organization and expression, thereby altering organismal phenotypes. However, their long-term evolutionary dynamics and effects on organismal fitness are often unknown. We addressed these questions by using the longest-running evolution experiment, during which twelve independent populations are propagated from a common E. coli ancestor in a glucose-limited environment for now over 60,000 generations (26 years). Most past studies have focused on point mutations and small InDels. Using evolved clones sampled over time in all 12 populations, we characterized all large-scale DNA rearrangements by using whole genome sequences and Whole Genome MappingTM (i.e optical mapping). After 40,000 generations, we identified a total of 110 rearrangements including 82 deletions, 19 inversions and 9 duplications. Many chromosomal regions were repeatedly affected by similar rearrangements and, at least in one population, they occurred early in evolution when fitness increase was strong. Therefore, many rearrangements may be under positive selection. At the very least, these rearrangements strongly affected the structure of the chromosome during evolution.At the molecular level, we showed that ~ 70% of all rearrangements occurred by recombination between Insertion Sequence (IS) elements, illustrating their importance in mediating genome plasticity. We therefore investigated the distribution and temporal dynamics of these small mobile genetic elements in all 12 populations. We showed that IS elements were strong contributors of the total mutations after 40,000 generations. In one population, they even represented about half of the total mutations and one IS type, IS150, revealed a strong 6-fold increase in copy number, accounting for the production of most of the rearrangements detected in this population. We showed that IS150 revealed a dynamic temporal behavior with a strong expansion followed by domestication by the host. By testing three evolutionary scenarios, we demonstrated that the IS150 expansion was related to a strong fitness increase conferred by the initial transposition events that occurred before 2000 generations. Later, between 20,000 and 40,000 generations, we measured a decreased transposition frequency, likely owing to a down regulation imposed by the host. Finally, and for the first time, we developed an evolution model of IS dynamics confirming that the IS expansion was related to a threshold number of initial IS beneficial insertions. All of our data showed that large-scale chromosomal rearrangements and IS elements have played an active role in the evolutionary outcomes after 40,000 generations of bacterial evolution
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Noll, Lance. "Detection and quantification of the top-seven Shiga toxin-producing Escherichia coli serogroups in feces and on hides of feedlot cattle and whole genome sequence-based analysis of O103 serogroup." Diss., Kansas State University, 2017. http://hdl.handle.net/2097/36204.
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Doctor of PhilosophyDepartment of Diagnostic Medicine/Pathobiology
Tiruvoor G. Nagaraja
Cattle are a reservoir for major Shiga toxin-producing Escherichia coli (STEC), which includes STEC O157 and the top six non-O157 serogroups (STEC-6; O26, O45, O103, O111, O121, O145). Collectively known as the STEC-7, these organisms are harbored in the hindgut and shed in the feces of cattle, which can contaminate hides. The de-hiding step during beef cattle processing can introduce fecal contaminants from the hide onto the carcass surface, creating the potential for contaminated beef products. The STEC-7 have been declared by the USDA-Food Safety and Inspection Service as adulterants in ground beef and non-intact beef products, and are monitored during beef cattle processing. However, many of the culture- and PCR-based tests for detection and/or quantification of the STEC, particularly of the STEC-6, are not established or require improvement and also virulence characteristics of STEC strains from cattle have not been fully analyzed. Therefore, the following studies were conducted: 1. Immunomagnetic separation (IMS)-based culture-method for detection of STEC-6 in cattle feces was developed and compared to a PCR-based method; 2. Detection sensitivity of pooled vs. individual IMS beads for isolation STEC-6 from cattle feces was evaluated; 3. Real-time PCR assay, based on the clustered regularly interspaced short palindromic repeat sequence polymorphisms (CRISPR), was developed and validated for serotype-specific detection and quantification of STEC O157:H7 in cattle feces; 4. Virulence gene profiles of bovine enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype E. coli O103 strains were examined with whole genome sequence (WGS)-based comparative analysis; 5. Prevalence and concentration of STEC-7 of fed-beef, cull beef and cull dairy cattle were determined. The culture and PCR methods detected all six serogroups in samples negative by the other method. Based on noninferiority tests, detection with pooled IMS beads was not inferior to detection with individual beads. Detection limits of the CRISPR-based qPCR assay for cattle feces spiked with pure cultures were 2.1 x 10³ and 2.3 x 10⁰ colony-forming units/g before and after enrichment, respectively. WGS-based analysis of E. coli O103 strains revealed key differences in the virulomes and mobilomes of EHEC, EPEC, and putative non-pathotype strains. The prevalence study revealed that a significantly higher (P < 0.01) proportion of hide samples from fed beef cattle (4.8%) were positive for STEC O157:H7, compared to samples from cull beef (1.6%) or cull dairy (0.2%); the majority of quantifiable STEC O157:H7 from each cattle type was at concentrations between 3 to 4 log CFU/100 cm². These data contribute to a knowledge gap on prevalence and concentration of STEC-7 and surrogate bacteria on cattle hides and carcasses, respectively. Furthermore, the development and refinement of culture- and PCR-based screening assays may lead to increased surveillance of major STEC serogroups, especially if the potential of WGS-based comparative genomics in identifying novel gene targets can be harnessed.
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Ghosh, Hiren [Verfasser]. "A genome-based approach to study the ecology and epidemiology of Extended-Spectrum Beta-Lactamases (ESBLs) producing E. coli / Hiren Ghosh." Gießen : Universitätsbibliothek, 2019. http://d-nb.info/1186624604/34.
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Stoesser, Nicole Elinor. "Applications of whole genome sequencing to understanding the mechanisms, evolution and transmission of antibiotic resistance in Escherichia coli and Klebsiella pneumonia." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:10ed1097-b2a1-4e3e-a4b3-58318d325f89.
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Whole genome sequencing (WGS) has transformed molecular infectious diseases epidemiology in the last five years, and represents a high resolution means by which to catalogue the genetic content and variation in bacterial pathogens. This thesis utilises WGS to enhance our understanding of antimicrobial resistance in two clinically important members of the Enterobacteriaceae family of bacteria, namely Escherichia coli and Klebsiella pneumoniae. These organisms cause a range of clinical infections globally, and are increasing in incidence. The rapid emergence of multi-drug resistance in association with infections caused by them represents a major threat to the effective management of a range of clinical conditions. The reliability of sequencing and bioinformatic methods in the analysis of E. coli and K. pneumoniae sequence data is assessed in chapter 4, and provides a context for the subsequent study chapters, investigating resistance genotype prediction, outbreak epidemiology in two different contexts, and population structure of an important global drug-resistant E. coli lineage, ST131 (5-8). In these, the advantages (and limitations) of short-read, high-throughput, WGS in defining resistance gene content, associated mobile genetic elements and host bacterial strains, and the relationships between them, are discussed. The overarching conclusion is that the dynamic between all the components of the genetic hierarchy involved in the transmission of important antimicrobial resistance elements is extremely complicated, and encompasses almost every imaginable scenario. Complete/near-complete assessment of the genetic content of both chromosomal and episomal components will be a prerequisite to understanding the evolution and spread of antimicrobial resistance in these organisms.
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Maistrenko, Oleksandr. "Variation in Core and Accessory Parts of Genome of Escherichia Coli Isolated from Soil from Riparian Areas in New York State." Thesis, North Dakota State University, 2016. https://hdl.handle.net/10365/28022.
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Escherichia coli is commensal bacteria and is a symbiont of the digestive system of vertebrates. Due to frequent deposition of E. coli into extrahost habitats (soil, water), approximately half of its population exists as free living organisms. It is unclear what genome-wide variation stands behind adaptation for extrahost habitat. This thesis applies a genome-wide association study approach to find genetic variation in core and accessory parts of genome of E. coli that is associated with 1) forest or agricultural field soil habitats and 2) with survival phenotype in soil microcosm. Gene composition analysis suggests that pan-genome of environmental E. coli is unlimited. Core and accessory genome contained variation associated with survival phenotype and with forest or field habitat.Federal Formula Funding (Hatch-Act)
ND-EPSCoR
Fulbright-STEP scholarship
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Sarica, Nazim. "Identification de contraintes locales impactant l’expression des gènes chez Escherichia coli." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL021.
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L'expression des génomes dépend de toutes les « contraintes » qui s'appliquent à l'ADN, que ce soit au niveau nucléotidique et génique (1D), qu'au niveau de son organisation conformationnelle (3D). Les études menées chez les bactéries ont montré que l'organisation 3D de leur chromosome circulaire s'échelonne du niveau moléculaire au niveau cellulaire. Au niveau moléculaire, les NAPs (Nucleoid Associated Proteins) organisent l'ADN en microdomaines d'environ 10 kb qui présentent des surenroulements de l'hélice d'ADN indépendants (Postow et al., 2004). Au niveau cellulaire, le chromosome est organisé en 4 régions structurées et 2 autres non structurées, appelées macrodomaines, d'une taille proche de 1Mb (Valens et al., 2004). Le projet consiste à étudier l'impact de contraintes topologiques sur la transcription des gènes en fonction de la position chez E. coli. La taille élevée du chromosome et les éléments interagissant avec rendent variable et dynamique l'organisation chromosomique. Il est donc difficile d'étudier certains aspects de la structure du chromosome et de corréler ces résultats avec l'expression géniqueA long list of constrains that affect gene expression have been discovered thanks to the thousands of sequenced genomes and comparative genomics. These contrains can be at the nucleotidic level, but also at the 3D scale. Studies showed that the spatial organization of bacterial genomes goes from the molecular level to the cellular level. At the molecular level, NAPs (Nuceloid Associated Proteins) are modeling the chromosome by inducing 10kb loops called microdomains, that present different supercoiling levels. At the cellular scale, it has been observed 4 different structured regions + 2 non-structured regions on E.coli's chromosome. These regions of approximatively 1Mbp are called macrodomains. The first goal of this project is to study the effects of positioning and constrains on gene expression in E.coli. With the chromosome being so large and precisely organized in time and space by several interacting elements simultaneously, it is often difficult for researchers to isolate one specific feature and to accurately correlate this to gene regulation. What becomes obvious when one begins to wade through the literature is that the field would greatly benefit from simplified model chromosomes
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Singh, Harminder. "Identification of Genes Encoding Acyl-Coa Reductases and Aldehyde Reductases in Mycobacterial Genome By Characterization of the Reductases Expressed in E. Coli." Master's thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3238.
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Mycobacterium tuberculosis has been long known to produce wax esters. However, the enzymes involved in their biosynthesis have not been identified. Here we report the identification of Rv3391 and Rv1543 as genes that encode fatty acyl-CoA reductases and Rv1544 as one that encodes an aldehyde reductase. When expressed in E.coli, the products of Rv3391 and Rv1543 catalyzed reduction of fatty acyl-CoA to fatty alcohol with the corresponding aldehyde as an intermediate with an optimal pH of 7.0. Both enzymes showed a strong preference for NADPH over NADH as a reductant. Apparent Km for NADPH was 38 [micro]M for Rv3391 product and 202 [micro]M for Rv1543 product. Both enzymes reduced saturated fatty acyl-CoA such as palmitoyl-CoA and stearyl-CoA but showed a preference for oleoyl-CoA. Apparent Km for oleoyl-CoA was 13 [micro]M for Rv3391 product and 7 [micro]M for Rv1543 product. The Rv1544 product catalyzed fatty aldehyde reduction to fatty alcohol but not acyl-CoA reduction. The optimal pH for aldehyde reduction was 8.0. This aldehyde reductase showed a strong preference for NADPH with an apparent Km of 83 [micro]M. All three reductases were inhibited by SH directed reagents.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
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Yesil, Mustafa. "Enhancing the inactivation of Escherichia coli O157:H7 by bacteriophage and gaseous ozone to improve postharvest fresh produce safety." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512103801957122.
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Launay, Adrien. "Etude de l'émergence de la diversité d'Escherichia coli in vivo par séquençage de génomes complets." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066692/document.
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Escherichia coli est une espèce commensale du tube digestif, mais elle peut aussi se révéler être un dangereux pathogène intra ou extra intestinal. Un même clone pouvant passer d'un état commensal à pathogène, la compréhension des mécanismes impliqués dans la diversification d'E. coli dans ces deux habitats représente un enjeu majeur de santé publique. Des expériences d'évolution expérimentale utilisant E. coli ont permis de révéler différentes facettes de l'adaptation bactérienne. Cependant, ces expériences de laboratoire utilisant des conditions artificielles, on peut s'interroger sur la pertinence des observations qui en découlent en milieu naturel et plus globalement s’interroger sur la part de la sélection naturelle dans la diversification de E. coli dans la nature. Pour répondre à ces questions, j'ai analysé les profils génomiques de diversification de E. coli au cours (1) d’une adaptation au tube digestif de souris ou (2) dans des infections extra-intestinales. Dans les deux cas, j’ai pu montrer une importante convergence au niveau du gène : un même gène étant muté plusieurs fois indépendamment, un signe que l’adaptation est active. Dans les infections aigues, des mutations touchant des régulateurs globaux ont été retrouvées, alors que dans le tube digestif les cibles de l’adaptation semblaient plus spécifiques. Enfin, les échantillons issus des infections incluant des souches a fort taux de mutation dites mutatrices, j'ai pu documenter pour la première fois la génomique de l'émergence de bactéries mutatrices en milieu naturel.En conclusion, mes travaux montrent que l’adaptation joue un rôle important dans la diversification de E. coli en milieu naturel et que ce processus s’apparente à celui observé dans des milieux artificiels de laboratoire. L’adaptation semble néanmoins plus active en conditions d’infections aigues que dans le tube digestif de sourisEscherichia coli is a commensal species living in the digestive tract of vertebrates, but can also be a harmful pathogen involved in both intra and extraintestinal diseases. As clones can behave both as commensals and pathogens, the comprehension of the mechanisms involved in the diversification of E. coli in those two habitats represents a major public health concern. In vitro experimental evolution studies using E. coli have unraveled the different faces of bacterial adaptation. However, as those experiments used artificial conditions, the relevance of these observations and more generally the contribution of adaptation to the diversification of E. coli in the wild remain questionable. To answer these questions, I analyzed the genomic profiles of diversification of E. coli during (1) adaptation to the mice digestive tract or (2) during acute extraintestinal infections. In both cases, I found a strong convergence at the gene level, i.e. observation of several impendent mutations in the same gene, suggesting a dynamic adaptation. In acute infections, mutations in global regulators were recovered, while more specific genes were recruited in the mice gut. Finally, the existence of clones with high mutation rate in the infections, allowed me to document for the first time the genomics of mutator emergence in the wild. In conclusion, my work shows that adaptation is playing an important role in the diversification of E. coli, and that this process is fairly similar to the one observed in the laboratory. Nevertheless, adaptation seems more active during infections than in the mice gut
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Mainda, Geoffrey. "Molecular epidemiology of antimicrobial resistance (AMR) and Shiga toxin producing E. coli (STEC) in dairy herds of central Zambia." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25416.
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Antimicrobial resistance (AMR) is a worldwide public health concern. While it is evident that the use of antibiotics creates selection pressure for the evolution of antibiotic resistance genes, there are still considerable knowledge gaps relating to the status quo of antibiotic use, emergence of resistant pathogens in different livestock production systems and spread within human and animal communities. This thesis includes a survey of antibiotic use in the dairy sector within a specific area of Zambia and analysis of AMR and virulence factors in E. coli isolated from dairy cattle and diarrhoea human patients with the following objectives. 1. To investigate the usage of antibiotics in the dairy sector and the drivers for use. 2. To determine the prevalence and patterns of antimicrobial resistance in E. coli isolated from faecal samples of dairy cattle. 3. To use whole genome sequencing (WGS) to investigate the molecular epidemiology of resistance determinants in E. coli strains isolated from both dairy cattle and humans. 4. To assess the zoonotic potential of isolated E. coli focusing on Shiga toxin-producing E. coli (STEC) and relationship to STEC associated with clinical disease in the UK. In view of these objectives, the first part of the work was carried out in Zambia and involved a questionnaire, a field survey, isolation of E. coli from dairy cattle faecal samples and phenotypic testing for AMR. In addition, E. coli isolates were obtained from another study that was focused on human patients presenting with diarrhoea at the University Teaching Hospital in Lusaka. The second part involved whole genome sequencing and molecular analyses of E. coli for resistance and virulence genotypes at the Roslin Institute (UK). For the field study, a stratified random sample of 104 farms was studied, representing approximately 20% of all dairy farms in the region. On each farm, faecal samples were collected from a random sample of animals and a standardised questionnaire on the usage of antibiotics was completed. An E. coli isolate was obtained from 98.67% (371/376) of the sampled animals and tested for resistance against the six types of antibiotics (tetracycline, ampicillin, sulfamethoxazole/trimethoprim, cefpodoxime, gentamicin and ciprofloxacin). These E. coli were then analysed together with those from humans for genotypes in the laboratory and from Illumina short read whole genome sequences using bioinformatics tools. Tetracylines and penicillin were the commonly used antibiotics in dairy herds. This finding was in line with the resistance phenotypes detected in E. coli isolated from the dairy cattle. The most prevalent AMR was to tetracycline (10.61; 95%CI: 7.40-13.82), followed by ampicillin (6.02; 95%CI: 3.31-8.73), sulfamethoxazole/ trimethoprim (4.49; 95%CI: 2.42-6.56), cefpodoxime (1.91; 95%CI: 0.46-3.36), gentamicin (0.89; 95%CI: 0.06-1.84) and ciprofloxacin (0%). The risk analysis indicated that AMR was associated with livestock diseases (lumpy skin disease and foot rot), exotic breeds (Jersey and Friesian), location, farm size and certain management practices. Analysis of whole genome sequences showed that isolates from humans had both higher levels and a greater diversity of resistance alleles than the cattle isolates. Common genotypes in both populations were: tetA (16%), tetB (10%), tetC (2%) for cattle isolates with tetA (32%), tetB (22%) and tetD (1%) in human isolates. Other common genotypes were blaTEM (56%), sul1 (29%), sul2 (66%), strA4 (57%) and strB1 (64%) in isolates of human origin while blaTEM (15%), sul1 (3%), sul2 (17%), strA4 (13%) and strB1 (19%) were in the cattle isolates. Whilst the E. coli isolates from cattle encoded resistance to common antibiotics of limited significance to human clinical medicine, isolates from humans had additional extended spectrum beta-lactamases (blaOXA, blaCMY, blaNDM, and blaDHA, blaOKP and blaCTX-M) that encode for resistance to essential antibiotics such as third generation cephalosporins and carbapenems. This was an evidence that AMR is an ongoing public health subject in Zambia but the exclusivity of certain resistances in the human population points to limited or no exchange of genotypes between E. coli of human origin and those from cattle. AMR in humans was probably independently selected by the use of antibiotics of clinical importance such as cephalosporin and fluoroquinolones. The virulence analysis focused on STEC, 11% (41/371) of E. coli isolates from cattle contained Shiga toxin genes (stx) while none (0/73) of the human isolates were positive. Phylogenetic analysis showed a random distribution of bovine STEC, with no indication of clonal spread. Although 89% (16/18) of the STEC tested had a cytotoxic effect on Vero cells, indicative of Shiga toxin production, only three (O45, O111, O157) belonged to one of the seven serogroups (O26, O157, O111, O103, O121, O145 and O45) associated with life-threatening enterohaemorrhagic E. coli (EHEC) infections in humans. In line with this, only the O157 serotype encoded a type 3 secretion system. This shows that, while Stx-encoding strains are common in these dairy herds of Zambia, they are not strain backgrounds known to pose an immediate threat to human health as they lack colonisation factors that are found in typical human EHEC. However, we must remain vigilant as emergence of EHEC strains in these animals remains an ever-present threat.
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Walz, Paul S. "Influence of pathogenic bacterial determinants on genome stability of exposed intestinal cells and of distal liver and spleen cells." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Biology, c2011, 2011. http://hdl.handle.net/10133/3405.
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Most bacterial infections can be correlated to contamination of consumables such as food and water. Upon contamination, boil water advisories have been ordered to ensure water is safe to consume, despite the evidence that heat-killed bacteria can induce genomic instability of exposed (intestine) and distal cells (liver and spleen). We hypothesize that exposure to components of heat-killed Escherichia coli O157:H7 will induce genomic instability within animal cells directly and indirectly exposed to these determinants. Mice were exposed to various components of dead bacteria such as DNA, RNA, protein or LPS as well as to whole heat-killed bacteria via drinking water. Here, we report that exposure to whole heat-killed bacteria and LPS resulted in significant alterations in the steady state RNA levels and in the levels of proteins involved in proliferation, DNA repair and DNA methylation. Exposure to whole heat-killed bacteria and their LPS components also leads to increased levels of DNA damage.xiv, 132 leaves : ill. (chiefly col.) ; 29 cm
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Royer, Guilhem. "Génomique comparée à grande échelle de souches d’Escherichia coli responsables de bactériémies chez l’Homme : implications cliniques et analyse des réseaux métaboliques PlaScope: a targeted approach to assess the plasmidome from genome assemblies at the species level Mortality in Escherichia coli bloodstream infections: antibiotic resistance still does not make it." Thesis, université Paris-Saclay, 2021. https://www.biblio.univ-evry.fr/theses/2021/interne/2021UPASL012.pdf.
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Escherichia coli est la bactérie aéro-anaérobie facultative majoritaire du tube digestif de l'Homme et, également, l'espèce la plus fréquemment isolée au cours de bactériémies dans les pays industrialisés. La population de E. coli présente une diversité importante mais néanmoins structurée, avec certains groupes phylogénétiques préférentiellement associés à un mode de vie (e.g. A/B1 et commensal, B2/D et pathogène extraintestinal). De nombreux facteurs de virulence ont été décrits chez les souches pathogènes extraintestinales (ExPEC), mais le pronostic des bactériémies à E. coli semble dépendre des facteurs associés à l'hôte. Cependant, certains clones virulents et multirésistants aux antibiotiques ont récemment émergé, modifiant drastiquement l'épidémiologie de ces infections. En parallèle, la démocratisation des méthodes de séquençage offre aujourd'hui une granularité encore jamais atteinte dans l'analyse des génomes bactériens. L'objectif de ce travail de thèse est de tirer profit des méthodes de génomique comparée pour améliorer notre compréhension de la physiopathologie des bactériémies à E. coli, tout en prenant en compte les modifications épidémiologiques majeures qui sont observées. La réalisation de telles comparaisons génomiques a nécessité, tout d'abord, la mise en place d'une stratégie d'analyse, incluant notamment le développement d'une approche ciblée pour l'identification des séquences plasmidiques au sein d'assemblages de génomes.Cette stratégie, appliquée à 545 souches recueillies au cours de l'étude Septicoli en 2016-2017, a permis de confirmer le rôle mineur des déterminants bactériens dans l'issue des bactériémies à E. coli. De plus, par la comparaison de ces souches à celles de l'étude Colibafi, nous avons étudié la dynamique de la population sur 12 ans. Si celle-ci apparaît globalement stable, l'exploration plus fine des principaux clones montre d'importants remaniements, parfois associés à des facteurs de virulence typiques des ExPEC. D'autre part, la diversité antigénique de ces clones est variable et suggère des pressions de sélection différentes en fonction de leur niche écologique respective. Enfin, dans une dernière partie nous avons réalisé la reconstruction des réseaux métaboliques de plus de 1400 souches de E. coli afin d'étudier les liens entre métabolisme et mode de vie. Les résultats soulignent la conservation du métabolisme chez E. coli et son association forte avec la phylogénie. Par ailleurs, une analyse détaillée des principaux clones retrouvés dans les bactériémies, dont notamment le ST131, met en évidence une voie métabolique impliquée dans la dégradation de composés aromatiques dérivés de la lignine. Cette voie, habituellement absente des souches de phylogroupe B2, pourrait procurer un avantage sélectif à ce clone pandémique mondial d'émergence récenteEscherichia coli is the predominant aero-anaerobic bacterium of the human gut and also the leading cause of bacteremia in industrialized countries. The E. coli population presents a high but structured diversity, with certain phylogenetic groups preferentially associated with a given lifestyle (e.g. A/B1 and commensal, B2/D and extraintestinal pathogen). Many virulence factors have been described in extraintestinal pathogenic strains (ExPEC), but the prognosis of E. coli bacteremia appears to be linked mainly to host-associated factors. However, some virulent and multi-resistant clones have recently emerged and dramatically modified the epidemiology of these infections. At the same time, the democratization of sequencing methods now offers a granularity yet never reached in the analysis of bacterial genomes. The objective of this thesis is to take advantage of comparative genomic methods to improve our understanding of the physiopathology of E. coli bacteremia, while taking into account the major epidemiological changes that are observed. Carrying out such genomic comparisons required, first of all, the implementation of an analysis strategy, including in particular the development of a targeted approach for the identification of plasmid sequences within genome assemblies.This strategy, applied on 545 strains collected during the Septicoli study in 2016-2017, confirmed the minor role of bacterial determinants in the outcome of E. coli bacteremia. In addition, by comparing these strains to those of the Colibafi study, we studied the population dynamics over 12 years. While the population appears to be stable overall, further exploration of the main clones shows significant changes, sometimes associated with virulence factors typical of ExPEC. On the other hand, the antigenic diversity of these clones is variable and suggests different selective pressures according to their respective ecological niches. Finally, in a last part we reconstructed the metabolic networks of more than 1400 E. coli strains in order to study the links between metabolism and lifestyle. The results highlight the conservation of metabolism in E. coli and its strong association with phylogeny. In addition, a detailed analysis of the main clones found in bacteremia, including the ST131, highlights a metabolic pathway involved in the degradation of aromatic compounds derived from lignin. This pathway, usually absent in phylogroup B2 strains, could provide a selective advantage to this recently emerging global pandemic clone
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Cavalcanti, Leonardo Sousa. "Papel da celulase XF-0818 na interação Xylella fastidiosa X Citros." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-16052005-162932/.
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A partir do sequenciamento do genoma da bactéria Xylella fastidiosa, agente causal da clorose variegada dos citros (CVC), estudos sobre os mecanismos de patogenicidade expressos durante a doença foram iniciados em projetos de genômica funcional, com o objetivo de confirmar a função das sequências gênicas identificadas no projeto Genoma de X. fastidiosa. Dentre esses mecanismos destacam-se as enzimas envolvidas no processo de ataque da bactéria. O presente trabalho teve como alvo de estudo a seqüência gênica denominada Xf-0818, clonada em Escherichia coli através da inserção em plasmídeo pET 28b(+), que presumivelmente codifica uma celulase de cerca de 60 kDa, possivelmente envolvida na degradação de fibras de celulose presentes nas membranas dos vasos pontuados do xilema de plantas de citros. A degradação desta membrana permitiria a movimentação de células bacterianas entre os vasos do xilema, o que é determinante para o desenvolvimento dos sintomas da CVC. A proteína foi superexpressada em E. coli por meio de indução com lactose e purificada através de ultrafiltração associada à cromatografia de afinidade a metal. A enzima apresentou alta atividade sobre a celulose carboximetilada 1%, avaliada através da quantificação de açúcares redutores. A obtenção de anticorpos foi realizada mediante injeção da enzima purificada em camundongos, os quais foram avaliados através de ELISA. A atividade da enzima sobre celulose carboximetilada foi reduzida após prévia incubação com os anticorpos. Esses anticorpos representam uma poderosa ferramenta em pesquisas visando à identificação das condições que favoreçam a expressão desta enzima in vivo. A presença de partículas de ouro coloidal em seções de tecido provenientes de plantas infectadas com X. fastidiosa indica a atuação da celulase durante a infecção, porém fazse necessária a confirmação do papel da proteína Xf-0818, através de novas análises com imunolocalização, além de estudos utilizando linhagens mutantes de X. fastidiosa para o gene que codifica esta celulase.From the genome sequencing of the bacteria Xylella fastidiosa, the agent responsible for the citrus variegated chlorosis (CVC), studies on the pathogenicity mechanisms expressed during the occurrence of the disease were started in genomic function projects, aiming to confirm the function of the genic sequences identified in the project X. fastidiosa Genome. Among these mechanisms, the enzymes involved in the bacterium attaching process are highlighted. The present study had, as a goal, the orf denominated Xf-0818, cloned in Escherichia coli by means of the insertion into pET 28b(+) plasmid, which codifies a cellulase of a size nearly 60 kDa, possibly involved in the degradation of cellulose fibers present in the membranes of the xylem vessels of citrus plants. The degradation of this membrane would allow for the movement of bacterial cells among the xylem vessels, which would be important for the development of CVC symptoms. The enzyme was over expressed into E. coli by means of their induction using lactose and purified using ultra-filtration associated to metal affinity chromatography. The enzyme presented high activity over 1%-carboximethyl cellulose (CMC), which was evaluated through quantification of released reduced sugars. The antibodies were obtained by injection of the purified enzyme into mice, which were evaluated by using the ELISA. The enzyme activity over CMC was reduced after previous incubation with the antibodies. These antibodies represent a tool for the researches aiming the identification of conditions that favor the expression of this enzyme in vivo. The presence of gold particles on the citrus diseased tissue samples indicate the participation of this cellulase on the disease, but other studies using immunocytochemistry and mutants of X. fastidiosa are necessary to confirm this hypothesis.
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Bürfent, Benedikt [Verfasser]. "The immunomodulatory capacity of helminths on inflammation: Impact of eosinophils on E. coli-induced sepsis and genome-wide transcriptome profiling of human monocytes stimulated with helminth extract and LPS implicate immune functions and diseases / Benedikt Bürfent." Bonn : Universitäts- und Landesbibliothek Bonn, 2017. http://d-nb.info/1149154284/34.
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Mendes, Renata de Siqueira. "Caracterização de proteínas de membrana de Leptospira interrogans expressas em Escherichia coli." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-27092011-151543/.
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O sequenciamento genômico da L. interrogans sorovar Copenhageni e os avanços das análises bioinformáticas permitiram a identificação de seis novos candidatos vacinais. Esses genes de Leptospira foram submetidos a ensaios de conservação do DNA genômico, RNA mensageiro e proteína nativa correspondente em doze sorovares de Leptospira, nos quais o gene LIC11469 foi o mais conservado. As proteínas recombinantes rLIC11469 e rLIC11030 foram purificadas por cromatografia de afinidade a metal, e submetidas a ensaio de dicroísmo circular. Em ensaios de localização celular pudemos observar a presença das proteínas nativas correspondentes na membrana externa de Leptospira. Ensaios de adesão mostraram a ligação da proteína rLIC11469 à laminina e ao plasminogênio. Ensaios de imunização e desafio demonstraram que a proteína rLIC11030 conferiu proteção contra infecção letal de L. interrogans em hamsters. Ambas as proteínas apresentaram reatividade com anticorpos presentes em soros de pacientes com leptospirose, sugerindo sua expressão durante a infecção.The genomic sequencing of the L. interrogans serovar Copenhageni and the advances of bioinformatics analysis allowed the identification of six new vaccine candidates. These genes were subjected to genomic DNA, mRNA and native protein conservation assays in twelve serovars from Leptospira, and the gene LIC11469 was the most conserved among these serovars. The recombinant proteins rLIC11469 and rLIC11030 were purified by metal affinity chromatography, and subjected to circular dichroism. Through cellular localization assays we could observe the presence of the corresponding native proteins on the outer membrane of Leptospira. In adhesion assays, the protein rLIC11469 showed binding to laminin and plasminogen. Immunization and challenge assays showed that the protein rLIC11030 afforded protection against lethal leptospiral inoculation in hamsters. Both proteins showed reactivity against sera of patients diagnosed with leptospirosis, suggesting that these proteins are probably expressed during infection.
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Souza, Natalie Michele de. "Avaliação da atividade imunogênica de três proteínas de Leptospira interrogans expressas em Escherichia coli." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-07062013-110750/.
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A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. No mundo, aproximadamente 500.000 casos são reportados a cada ano, com 10% de taxa de mortalidade. Atualmente, vacinas contra leptospirose são compostas por células inativadas e são ineficazes em diferentes aspectos. Após analise do genoma, os genes LIC11121, LIC11087, LIC11228 e LIC11084 foram escolhidos para caracterização da imunogenicidade de suas respectivas proteínas. Esses genes foram clonados no vetor de expressão pAE e as proteínas recombinantes foram purificadas. Os resultados sugerem que essas proteínas podem estar localizadas na membrana externa, são imunogênicas, possivelmente expressas durante a infecção e que podem ter envolvimento em mecanismos de evasão do sistema imune e de patogenicidade da bactéria. Além disso, em um de dois experimentos, a proteína rLIC11084 induziu imunidade protetora parcial em hamsters imunizados frente desafio letal.Leptospirosis is a zoonotic disease caused by pathogenic bacteria of genus Leptospira. In the world, nearly 500,000 cases are reported each year, with 10% of mortality rate. Currently, vaccines against leptospirosis are composed by inactivated cells that are ineffective in many aspects. After genome analysis, the genes LIC11121, LIC11087, LIC11228 e LIC11084 were chosen for immunogenicity characterization of their respective proteins. These genes were cloned in the pAE expression vector and the proteins encoded by LIC11087, LIC11228 and LIC11084 were purified. The results suggest the localization of these proteins in the bacterial outer membrane, are immunogenic, are possibly expressed during infection and may have involvement in mechanisms of immune system evasion and pathogenicity. Moreover, in one of two experiments, the rLIC11084 protein induced partial protective immunity of immunized hamsters against lethal challenge.
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Davenport, Colin [Verfasser], and Burkhard [Akademischer Betreuer] Tümmler. "Genomic and metagenomic application of microbial genome signatures / Colin Davenport. Burkhard Tümmler. Biomedical Research School (HBRS) Department of Paediatrics Hannover Medical School." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2010. http://d-nb.info/100117173X/34.
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Walters, D. E. "A further nar gene in Escherichia coli." Thesis, University of Leeds, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383761.
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Chan, Kamfai. "Erythromycin Susceptibility and Genomic Regions Characterization of Campylobacter coli." NCSU, 2007. http://www.lib.ncsu.edu/theses/available/etd-05162007-114033/.
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Campylobacter jejuni and Campylobacter coli are major bacterial food-borne pathogens that cause enteric diseases worldwide, resulting in significant public health and economic burden. These two closely related species colonize a wide range of farm animals including turkeys, pigs, chickens, and cattle. Consumption of meat (especially poultry) contaminated with C. jejuni or C. coli has been implicated as the major route of infection. When needed, antibiotics used for treatment are fluoroquinolones or macrolides such as erythromycin. Recent studies have shown that the percentage of C. coli that have resistance to antimicrobials, including those typically used for treatment of human campylobacteriosis, has increased, making it a top priority to investigate the mechanisms for acquisition and dissemination of antimicrobial resistance in these pathogens. Certain Campylobacter strains harbor a transcribed intervening sequence (IVS) in their 23S rRNA genes. Following transcription, the IVS is excised, leading to fragmentation of the 23S rRNA. The origin and possible functions of the IVS are unknown. Furthermore, the distribution of IVS-harboring strains within Campylobacter populations is poorly understood. In this study, strains of Campylobacter coli from turkeys, representing numerous different multilocus sequence typing (MLST)-based sequence types (STs), were characterized in terms of IVS content and erythromycin susceptibility. We identified strains that harbored IVSs in all three 23S rRNA genes, as well as strains lacked IVSs from at least one of the genes. The STs of the latter strains belonged to an unusual cluster of C. coli STs (?cluster II?), earlier found primarily in turkey strains, and characterized by presence of the C. jejuni aspA103 allele. The majority of strains harboring IVSs in all three 23S rRNA genes were resistant to erythromycin. In contrast, cluster II strains, which harbored at least one IVS-free 23rRNA gene, were susceptible to the antibiotic. Cluster II strains could be transformed to erythromycin resistance with genomic DNA from C. coli that harbored IVS and the A2075G transition in the 23S rRNA gene, associated with resistance to erythromycin in Campylobacter. Erythromycin-resistant transformants harbored both the A2075 transition and IVS. The findings suggest that absence of IVS in C. coli from turkeys is characteristic of a unique clonal group of erythromycin-susceptible strains, and that IVS can be acquired by these strains via natural transformation to erythromycin resistance. Analysis of C. coli and C. jejuni strains isolated from broilers, turkeys and swine has shown the association of the lack of IVS and erythromycin susceptibility is unique to C. coli from turkeys. The presence of the C. jejuni aspA103 allele in the chromosome of cluster II C. coli strains is a unique characteristic of this clonal group. To further characterize the genome composition in the aspA region in cluster II strains, we determined the corresponding DNA sequences from two turkey-derived cluster II C. coli strains (6979 and 7474, of ST-1150 and ST-1161, respectively). Genomic organization upstream of the aspA gene was divergent between these two cluster II strains and the reference strain C. coli RM2228, the genome sequence of which has been completed. Genes encoding a putative Crp-family transcriptional regulator (CCO0137) and a conserved hypothetical protein (CCO0138) that were present in C. coli RM2228 and C. coli 6818 were not found in the same genomic region in C. coli 6979 or C. coli 7474. Moreover, single nucleotide polymorphism (SNP) analysis revealed that genes encoding subunit II of cytochrome d ubiquinol oxidase (cydB) and a putative aspartate racemase (CJ0085c) harbored numerous C. jejuni-specific SNPs. Interestingly, genes encoding subunit I of cytochrome d ubiquinol oxidase (cydA) and uracil-DNA glycosylase (ung) harbored C. coli-specific SNPs in their 5? portions but C. jejuni-specific SNPs in their 3? portions, suggesting that these may be hybrid genes that were originated from C. jejuni and C. coli. Our data suggest the presence of recombination events in the genomic region between cydA and ung in cluster II strains of C. coli. Such genomic features may contribute to the observed prevalence of cluster II strains among C. coli from turkeys, and to the characteristic susceptibility to erythromycin exhibited by these strains.
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Sun, Xueguang. "Characterization of E. coli HFQ structure and its RNA binding properties." Available online, Georgia Institute of Technology, 2006, 2006. http://etd.gatech.edu/theses/available/etd-12052005-145342/.
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Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2006.Wartell Roger, Committee Chair ; Chernoff Yury, Committee Member ; Harvey Stephen, Committee Member ; Spiro Stephen, Committee Member ; Williams Loren, Committee Member.
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Price, Erin Peta. "Development of novel combinatorial methods for genotyping the common foodborne pathogen Campylobacter jejuni." Thesis, Queensland University of Technology, 2007. https://eprints.qut.edu.au/16601/1/Erin_Peta_Price_Thesis.pdf.
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Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni.
Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR.
Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method.
The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis.
Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs".
Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.
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Price, Erin Peta. "Development of novel combinatorial methods for genotyping the common foodborne pathogen Campylobacter jejuni." Queensland University of Technology, 2007. http://eprints.qut.edu.au/16601/.
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Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.
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Mahmoud, Nada. "Next-generation diagnostics of escherichia coli from community-onset sepsis patients in sweden : Studying the biodiversity of escherichia coli genomes." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-19893.
Full textAbstract:
Escherichia coli (E. coli) is among the gram-negative bacteria that can cause several infections including sepsis. Confirmed sepsis patients must show a sequential organ failure assessment (SOFA) score of ≥2 with verified infection. Understanding the genotypic characteristics of E. coliclinical isolates from sepsis patients can help directing treatment strategies, tracking antibiotic resistance, and monitoring acquired virulence factors that can contribute to the severity of sepsis. The isolates included in this thesis were collected from confirmed sepsis patients (SOFA 2-3)during a prospective observational study that was conducted in Sweden. The aim was to study the biodiversity in E. coli clinical isolates using whole genome sequencing (WGS) paired-end reads that were produced by the next-generation sequencer Illumina. To perform the WGS-based analysis, two bioinformatics pipelines were used. The first is the in-house developed pipeline and the second is the 1928Diagnostics E. coli pipeline. The obtained in silico results were compared with the phenotypic findings for species identification and the in vitro predictions of antibiotic resistance. Species identification by the bioinformatics pipelines matched the phenotypic method, except for three isolates that were highly contaminated with other species. Both pipelines predicted the exact multi locus sequence types, which revealed that the most common sequence types (STs) were ST73(17%), ST95(9%), and ST131(6%). The phenotype of the isolates resulted in 5% resistant to at least one of the assessed antibiotics. The 1928Diagnostics predicted 28% of the isolates were resistant to at least one class of the tested antibiotic classes, while the in-house pipeline predicted 33% of the isolates to be resistant. Out of the predicted resistant isolates, 52% coded for multi-drug resistance. The in-house pipeline reported virulence genes. The common reported genes were coding for iron reuptake, adhesins, cell outer membrane and increased serum survival. It was observed that the isolates that belonged to ST73 and ST95 showed a more susceptible antibiotic profile than isolates that belonged to ST131, those harbored the highest mean of virulence genes. In conclusion, the present study provided an evidence of the usefulness of the WGS-based analysis to study the biodiversity in E. coli. The obtained results are valuable for surveillances, tracking antibiotic resistance and identifying virulence factors, but with a limited use in clinical settings.
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Santos, Ana Carolina da Silva. "Caracterização fenotípica, sequenciamento e anotação do genoma total de uma cepa de Escherichia coli isolada de uma paciente com Doença de Crohn." Botucatu, 2015. http://hdl.handle.net/11449/144081.
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Orientador: Josias RodriguesBanca: Silvio Luís de Oliveira
Banca: Rogéria Keller
Resumo: A Doença de Crohn (DC) é uma variante das doenças inflamatórias intestinais que não tem uma causa definida, porém há o envolvimento de fatores ambientais e genéticos, entre eles a microbiota. Portadores da DC apresentam desequilíbrio na composição de espécies da microbiota intestinal, incluindo o aumento da Escherichia coli. A E. coli é uma bactéria versátil em sua relação com o hospedeiro, isto é, inclui cepas comensais e várias categorias patogênicas. Estas compreendem dois grupos: linhagens causadoras de infecções intestinais e linhagens causadoras de infecção extraintestinais. As E. coli isoladas de portadores da DC em geral pertencem ao segundo grupo, e uma das principais características apresentadas por estas bactérias é a interação com células epiteliais (aderência e invasão). Estudos feitos na década de 90 levaram a identificação de um novo patótipo de E. coli, capaz de aderir e invadir células epiteliais e ainda invadir e se replicar dentro de macrófagos, produzindo granulomas, característica histopatológica da DC. Este novo patótipo, chamado de Adherent and Invasive E. coli (AIEC), têm sido usada como referência na caracterização de E. coli isoladas a partir de portadores da DC. Neste trabalho, foi feito o estudo de caracterização fenotípica e genética de E. coli isoladas a partir de 9 portadores da DC e 5 pacientes controles, as quais foram submetidas a testes de adesão e invasão celular em células epiteliais, teste de formação de biofilme e identificação de filogrupos da coleção de referência de E. coli (EcoR). Dentre o conjunto de amostras bacterianas estudado, foi encontrada uma amostra isolada de uma paciente com DC que apresentou invasibilidade superior às demais, e foi intensivamente estudada, tendo-se verificado que ela apresenta um perfil de virulência distinto de AIEC. O genoma da amostra em questão foi sequenciado e anotado. Os resultados da caracterização...
Abstract: Not available
Mestre
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Mahfouz, Norhan, Serena Caucci, Eric Achatz, Torsten Semmler, Sebastian Guenther, Thomas U. Berendonk, and Michael Schroeder. "High genomic diversity of multi-drug resistant wastewater Escherichia coli." Nature Publishing Group, 2018. https://tud.qucosa.de/id/qucosa%3A32482.
Full textAbstract:
Wastewater treatment plants play an important role in the emergence of antibiotic resistance. They provide a hot spot for exchange of resistance within and between species. Here, we analyse and quantify the genomic diversity of the indicator Escherichia coli in a German wastewater treatment plant and we relate it to isolates’ antibiotic resistance. Our results show a surprisingly large pan-genome, which mirrors how rich an environment a treatment plant is. We link the genomic analysis to a phenotypic resistance screen and pinpoint genomic hot spots, which correlate with a resistance phenotype. Besides well-known resistance genes, this forward genomics approach generates many novel genes, which correlated with resistance and which are partly completely unknown. A surprising overall finding of our analyses is that we do not see any difference in resistance and pan genome size between isolates taken from the inflow of the treatment plant and from the outflow. This means that while treatment plants reduce the amount of bacteria released into the environment, they do not reduce the potential for antibiotic resistance of these bacteria.
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Sun, Xueguang. "Characterization of E coli Hfq structure and its RNA binding properties." Diss., Georgia Institute of Technology, 2005. http://hdl.handle.net/1853/10416.
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Hfq is a bacterial RNA-binding protein recently shown to contain the Sm motif, a characteristic of Sm proteins that function in RNA processing in archaea and eukaryotes. Hfq plays a major role in RNA-RNA interactions regulating translation. Comparative structural modeling and amino acid sequence alignment were used to predict the 3-D structure of Hfq and the model was in excellent agreement with the crystal structure which determined for S. aureus Hfq. The evolution of Hfq was explored by a BLAST search of microbial genomes followed by phyletic analysis. About half of the genomes examined contain at least one gene coding for Hfq. The presence and absence of Hfq closely followed major bacterial clades. The potential RNA binding residues on the two surfaces of the Hfq hexamer were proposed based on the bioinformatics studies and the mutant Hfq proteins with either single or double mutations on the two surfaces of the Hfq hexamer were generated. Their RNA binding properties was biophysically studied by gel-shift assay, fluorescence anisotropy and fluorescence quenching techniques. Results indicated that 1) point mutations on the distal surface of the Hfq hexamer, Y25A and K31A, have a major effect on A18 binding. Both reduce binding by about 1000 fold. Mutations on the proximal surface have a small or no influence on A18 binding. 2) Two mutations, F39A and R16A, on the proximal surface of the Hfq structure reduce binding to the DsrA domain II by 10 fold. Other mutations reduce binding by less than 2 fold. 3) An amino acid covariance was observed in L12 and F39. Mutation L12F can partially restore F39A in DsrA RNA binding. 4) It appears that two Hfq hexamers cooperatively bind one RNA for both DsrADII and A18.
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Bin, Thani Ali Salman. "Genomic diversity of ten Escherichia coli strains associated with bloodstream infections." Thesis, University of Leicester, 2009. http://hdl.handle.net/2381/4246.
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Escherichia coli are usually regarded as a harmless human colonic flora. However, pathogenic strains of E. coli have been associated with infections that could range from infected mucosal surfaces by intestinal pathogenic E. coli to the more severe cases of disseminated infections throughout the body by the extraintestinal pathogroups. The main focus of this project was to investigate the genomic contents of pathogenic bloodstream infection (BSI)-associated E. coli strains. This is because the genome contents of the E. coli BSI-associated isolates have not been well studied, with only few reports indicating that the pathogenincity of these strains could be attributed to horizontally acquired DNAs known as genomic islands (GEIs). The genomic contents of 10 clinical BSI-associated E. coli strains, isolated at the Leicester Royal Infirmary were investigated in this study. The first approach used to investigate the genomic contents of these strains was by interrogating the downstream ends of tRNA genes for their GEI contents by the sequential PCR strategy tRIP-PCR (tRNA interrogation for pathogenicity islands) followed by the SGSP-PCR (single genome specific primer-PCR). In this approach the flanking regions of the tRNA sites were used to first screen the tRNA genes for their GEIs followed by amplifying the boundaries of the identified GEIs. In the second approach termed Microarray-Assisted mobilome Prospecting (MAmP), the physical genome size of the tested strains obtained by the pulsed-field gel electrophoresis (PFGE) is compared to the sum total of the bits of the genome detected or visualized by the array. The difference between the two measurements is used to estimate the size of the novel, non-microarray-represented mobile genome (mobilome) present in the tested strains. Remarkably, despite only studying 10 E. coli strains, associated with a single disease type the tRIP-PCR method has identified at least 3 GEIs that contain novel sequences, and 46 GEIs, resembling uropathogenic E. coli CFT073-like entities. One particular strain E105 had 13 tRNA sites occupied with GEIs. On the other hand, an average novel, non-microarray-borne mobilome of (219 kb /strain) was obtained by the MAmP which, corresponds with previous studies. The strategies used in this study had proved successful in addressing and identifying mobilome-rich strains. Therefore, using such approaches in combination with whole genome sequencing progects could prioritize the strains and the genomic regions that need to be sequenced. Such prioritization would avoid sequencing of hundreds of isolates to identify their novel gene pool and would reduce the cost of genomic sequencing. Moreover, applying such approaches for the identification of new virulence genes and/or pathogenic mechanisms could lead to significant improvements in the treatment of E. coli infections.
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Eger, Nicole. "Advanced Detection Methods of Genomic Barcodes for Genotyping Escherichia coli Libraries." Thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-439038.
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Pooled cell strain libraries are a powerful tool allowing to investigate the influence of genetic modifications on phenotypes in high throughput single-cell assays. To link the genotype to phenotype in each cell of the library, unique 20 base pairs (bp) long barcodes are used to allow in situ genotyping after phenotyping via fluorescence microscopy. In previous studies, these barcode sequences were expressed from high copy number plasmids resulting in a high number of targets for detection via fluorescence in situ hybridization (FISH) and thus, a strong readout signal. However, constant selection pressure must be applied on the cells to maintain the foreign plasmid DNA which may influence the phenotype. Inserting unique barcodes on the chromosome ensures stability of the construct which is required for some genomic library applications. However, the low copy number of the barcode sequence often requires an additional step of DNA amplification for efficient detection. In this study, two methods for barcode amplification were investigated. First, amplification from the double stranded DNA upon binding of peptide nucleic acids and subsequent amplification via rolling circle amplification (AmPPR). Second, amplification from genomic DNA or cDNA via loop-mediated isothermal amplification (LAMP). Whereas the AmPPR approach remained unsuccessful, chromosomal barcode sequences were successfully amplified in situ via LAMP and subsequently detected using FISH. I show that LAMP can potentially be a quick, specific, and elegant amplification technique for in situ genotyping in microfluidic devices. However, nonspecific amplification and partly nonspecific readout signals when using LAMP remain a problem and need to be further investigated before implementing this method on pooled libraries.
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Nelson, Adam Michael. "Genomic analysis of pathogen evolution virulence gene acquisition and genetic erosion in Escherichia coli /." Diss., Connect to online resource - MSU authorized users, 2008.
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